Introduction
Coldwater disease (CWD) is a worldwide problem in
commercial (foodfish) aquaculture as well as public resource
enhancement hatcheries, and is caused by the gramnegative
bacterium Flavobacterium psychrophilum. Juvenile
trout and salmon infected with this disease experience high
mortalities, and surviving fish often exhibit skeletal deformities
associated with spinal compression. Aquaculture vaccines
have become important tools in preventing fish diseases,
but a commercial vaccine is not currently available for CWD.
Development in this area has likely been hampered by
altered expression of virulence factors among different
strains of F. psychrophilum and an incomplete understanding
of the immune response of fish to this pathogen. It has
been noted that protease differences exist between strains
(Dalsgaard 1993, Bertolini et al. 1994, Madsen and
Dalsgaard 1999), but the identities of these components and
roles they play in the fish immune response are unknown.
Significant protection has been observed under laboratory
conditions following immunization with F. psychrophilum
(Holt 1988, Obach and Laurencin 1991), but antigenicity
appears to be strain dependent.

A study is currently underway in our laboratory using twodimensional
polyacrylamide gel electrophoresis (2-D PAGE) to identify antigens and
virulence factors of this bacterium that may be targeted for the development
of an efficacious vaccine for the aquaculture industry. 2-D gel profiles
of virulent and nonvirulent strains are being compared to iden
tify and
further characterize antigens of interest. The role these play in pathogen
virulence and immunoprotection is currently being evaluated using the
PROTEAN IEF cell and ReadyStripTM IPG strips to isolate and excise specific
bacterial proteins. Sequential extraction procedures are being utilized
along with biotin labeling to distinguish between cytoplasmic, membrane-associated,
and cell surface proteins. Since fish are capable of mounting a protective
immune response against F. psychrophilum, it is speculated that a 2-D
approach will be valuable in allowing identification and characterization
of immunodominant and virulence-associated proteins in relation to specific
bacterial strains.

MethodsBacterial Culture
Virulent (CSF-259-93) and nonvirulent (ATCC 49418) isolates of F. psychrophilum
were cultured in tryptone yeast extract salts broth at 15C. Bacteria
used for 2-D PAGE were harvested from log-phase cultures and adjusted
to an absorbance of 0.4 at 525 nm. This corresponds to an approximate
bacterial concentration of 5 x 107 cfu/ml.

Biotinylation of Cell Surface Components
In order to detect cell surface membrane proteins and allow comparison
to sequentially extracted proteins, viable bacteria were biotinylated
prior to protein extraction. Bacteria were harvested in log-phase growth
at a concentration of approximately 5.0 x 107 viable bacteria/ml (0.4
absorbance) and washed twice in ice-cold PBS (pH 8.0) taking care not
to disrupt or lyse cells. The bacteria were resuspended to an absorbance
of 0.2 at 525 nm and 1.7 mg of sulfo-NHS-LC-biotin was added per ml of
bacteria. This mixture was allowed to rock for 30 min at room temperature.
The bacteria were then wa
shed in ice-cold PBS and resuspended to an approximate
protein concentration of 12 mg/ml in reagent 1. Washed cells were subjected
to the initial extraction step (see above) and an equal volume of reagent
3 was added to obtain total proteins for 2-D PAGE separation. Following
2-D PAGE and transfer of separated proteins to a nitrocellulose membrane,
biotin-labeled proteins were detected by probing blots with ExtrAvidin-alkaline
phosphatase (Sigma) and visualized colorimetrically by the addition of
NBT/BCIP.

IEF and Second-Dimension Separation of Proteins
Sequentially extracted or total protein samples were applied to ReadyStrip
IPG Strips pH 47 or pH 310 and passively rehydrated overnight in a humidity
chamber. Once fully rehydrated, isoelectric focusing of proteins was carried
out with the PROTEAN IEF cell using a preset method that allowed a minimum
of 20,000 V-hr to be obtained while maintaining a constant temperature
of 17C. Focused strips were stored at -80C until second-dimension PAGE
could be performed.

ResultsSequential Extraction and Biotin Labeling of Bacterial Proteins
Using the CSF-259-93 reference strain, th
e ability to separate cellular
proteins based on their hydrophobicity was examined. The relationship
of extracted proteins to cell surface proteins was then determined by
comparison of 2-D gel profiles of biotin-labeled proteins. The results
clearly demonstrate the ability of the extraction procedure to isolate
proteins associated with cytoplasmic or cell membrane compartments of
the bacteria (Figure 1).

Comparison of gel profiles shows that a large number of proteins are
extracted in the E1 cell lysate fraction. Membrane-associated (less soluble)
fractions were then obtained following E2 and E3 separation. Subsequent
comparison to biotin-labeled proteins (Figure 2) shows that a dominant
string of proteins (circled) at an approximate molecular weight of 100
kD and pI values between 5.2 and 5.8 represents a cell surface component
and appears to primarily correspond to proteins in extraction E2 of Figure
1. Additional biotin-labeled proteins are present in the 20 kD range (pI
approximately 5.15.6) but are not readily identified in the sequentially
extracted fractions.

Comparison of Total Proteins from Virulent and Nonvirulent Strains of
F. psychrophilum
Separation and comparison by 2-D PAGE of total proteins over the pH 310
range from strain CSF-259-93 (virulent) and ATCC 49418 (nonvirulent) demonstrate
that proteins are differentially expressed between bacterial strains (Figure
3). Initial qualitative analysis reveals that the majority of bacterial
proteins separate in the molecular weight ranges of approximately 40150
kD and have pI ranges of approximately 4.56.5. It appears that the nonvirulent
ATCC strain has a higher concentration of proteins in this rang
e, but
distinct proteins at lower molecular weight did not resolve as well or
were less concentrated in the ATCC strain than the CSF strain. Two major
proteins (p15 and p53) are represented in the virulent (CSF) strain but
do not appear in the ATCC strain, while at least one protein (p37) appears
to be differentially expressed in the ATCC strain.

Conclusions
The techniques outlined here demonstrate the effectiveness of sequential
extraction in obtaining bacterial proteins from different compartments
of the cell. Combined with 2-D PAGE techniques, the ReadyPrep sequential
extraction kit allowed cell-associated and cytoplasmic proteins to be
effectively separated. In addition to the development of techniques to
separate different bacterial protein components, we have successfully
separated proteins from both virulent and nonvirulent strains of F. psychrophilum
by 2-D PAGE. The use of the PROTEAN IEF cell allowed rapid throughput
of samples and exceptional control over focusing conditions. The separation
of distinct proteins using these methods has allowed the comparison of
potential virulence factors between two bacterial isolates grown under
identical conditions. Further characterization is needed and will focus
on comparison of differentially expressed proteins to active proteases
(potential virulence components) and immunodominant bacterial antigens.
Protein sequencing and eventual gene isolation may lead to the development
of an efficacious recombinant vaccine for aquaculture, or may allow screening
of the many F. psychrophilum isolates for potential vaccine candidates.
This approach has the potential to substantially advance our current understanding
of
this pathogen and may lead to better methods of disease control.

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